www.fluorescencemicroscopy.it

Fluorescence

Some materials when are excited by short wavelength light emitt light of wavelength higher. This phenomenon is called fluorescence. The fluorescence is divided into primary and secondary. When a substance emits flurescenza individually, after being excited by light at a certain wavelength, it is called primary fluorescence, a substance emitting primary fluorescence is the chlorophyll. More often it is secondary or induced fluorescence that is produced by a particular type of histological staining called "fluorochrome", it is used to "mark" the specific material to be analyzed.
The light source used to irradiate the subject is high-energy ( HBO or XENON lamps) and short wavelength (eg UV or blue), the emitted light is low energy and longer wavelength (eg green or red) under the law of Stokes.
In the phenomena of fluorescence, intensity of the light is specific for each substance and decreases with time very quickly, with an exponential law similar to the radioactive decay, in fact, we can say that the fluorescence, unlike phosphorescence, ceases to end of the cause of excitement.
To view the phenomenon of fluorescence requires a special type of microscope: the fluorescence microscopy.
The fluorescence microscope was developed by Köhler, Rechert and Lehman in the early 20th century, but only after a few decades it was possible to see its full realization.
In this type of microscope is placed in the beam path a filter system, excitation, dichroic and barrier.
The excitation filter to illuminate the sample with selected wavelengths from the source. The barrier filters are chosen to block (absorb) the wavelengths of excitation and allow to pass only the wavelengths emitted from the sample.
The dichroic mirrors are specialized filters designed to efficiently reflect the excitation wavelength and let the wavelength of emission.
The correct choice of these filters is critical in fluorescence microscopy.
There are two types of fluorescence microscopy, transmitted light or incident light. In the following pages both techniques are described in detail.